smear bands in western blot

Hello to everybody. It is a pleasure joining such a marvellous community!

I have a persisting problem with my western-blots which is really annoying me. I always obtain a smear in the lines of my westerns and I do not know where they come from. It starts in the wells and go down until the middle of the membrane, approximately. I have tried several variations:

1. I tried two different buffers to isolate proteins, one contains SDS and DTT, while the other contains glycin, triton and beta-mercapto.
2. I have varied the quantity of proteins loaded, from 10 to 50 micrograms. I always get the same smear.
3. I also varied the concentration of first antibody (from 1:2000 to 1:20000) and the stringency of washes

With all these variations I could observed difference in the band pattern (I mean, below the smear), but the smear is always the same. The only idea I have is that maybe I have a conamination with glucids, but I do not know whether this is possible and, if so, what can be done.

Hello to everybody. It is a pleasure joining such a marvellous community!

I have a persisting problem with my western-blots which is really annoying me. I always obtain a smear in the lines of my westerns and I do not know where they come from. It starts in the wells and go down until the middle of the membrane, approximately. I have tried several variations:

1. I tried two different buffers to isolate proteins, one contains SDS and DTT, while the other contains glycin, triton and beta-mercapto.2. I have varied the quantity of proteins loaded, from 10 to 50 micrograms. I always get the same smear.3. I also varied the concentration of first antibody (from 1:2000 to 1:20000) and the stringency of washes

With all these variations I could observed difference in the band pattern (I mean, below the smear), but the smear is always the same. The only idea I have is that maybe I have a conamination with glucids, but I do not know whether this is possible and, if so, what can be done.

May anyone help me, please?

Smearing would most likely be caused during the SDS-PAGE step. Could be an indicator that total protein wasn't separated evenly? What are your SDS-PAGE settings?

I've been doing alot of westerns recently and haven't had much experience prior to this so I really think this forum is useful in sharing and troubleshooting.How do the 10, 20 and 40 look vs 50 ug?

There seems to be a faint band around the 32kDa size (in the forth lane sample from left and last lane) - your band should appear in between the 3rd and 4th last ladder (25-35 kDa region). But I seriously haven't seen such a smear before. Try reducing the voltage of your SDS-PAGE to maybe 90 or 100V. Did you try to stain for a housekeeping protein (e.g. actin, tubulin, GAPDH)? Might be worth doing that to see if those are even present.

If your protein of interest isn't a large one (falling in the bad smear region), I would not worry as much because the bottom half is pretty clean and any potential band should turn up.

I am going to use a E. coli extract with a protein containing the same tag overexpressed to check whether the smear appears and also whether I can clearly detect an overexpressed protein. I´ll try less voltage, following your piece of advise.